Vitamin E Analog Trolox Attenuates MPTP-Induced Parkinson’s Disease in Mice, Mitigating Oxidative Stress, Neuroinflammation, and Motor Impairment

Trolox is a potent antioxidant and a water-soluble analog of vitamin E. It has been used in scientific studies to examine oxidative stress and its impact on biological systems. Trolox has been shown to have a neuroprotective effect against ischemia and IL-1β-mediated neurodegeneration. In this study, we investigated the potential protective mechanisms of Trolox against a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson’s disease mouse model. Western blotting, immunofluorescence staining, and ROS/LPO assays were performed to investigate the role of trolox against neuroinflammation, the oxidative stress mediated by MPTP in the Parkinson’s disease (PD) mouse model (wild-type mice (C57BL/6N), eight weeks old, average body weight 25–30 g). Our study showed that MPTP increased the expression of α-synuclein, decreased tyrosine hydroxylase (TH) and dopamine transporter (DAT) levels in the striatum and substantia nigra pars compacta (SNpc), and impaired motor function. However, Trolox treatment significantly reversed these PD-like pathologies. Furthermore, Trolox treatment reduced oxidative stress by increasing the expression of nuclear factor erythroid-2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1). Lastly, Trolox treatment inhibited the activated astrocytes (GFAP) and microglia (Iba-1), also reducing phosphorylated nuclear factor-κB, (p-NF-κB) and tumor necrosis factor-alpha (TNF-α) in the PD mouse brain. Overall, our study demonstrated that Trolox may exert neuroprotection on dopaminergic neurons against MPTP-induced oxidative stress, neuroinflammation, motor dysfunction, and neurodegeneration.


Introduction
Parkinson's disease (PD), first recognized by James Parkinson in 1817, is the second most common neurodegenerative disease. It affects 1.5% to 2.0% of individuals older than 60 years and 4% of people over 80 years of age [1,2]. Primarily, it is a movement disorder and clinically, it is characterized by resting tremors, postural instability, bradykinesia, slurred speech, and rigidity [3]. PD is a heterogeneous disorder with a complex etiology. It is believed to begin with the degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc) [4]. In PD mitochondrial dysfunction, protein misfolding, oxidative stress, and neuroinflammation occur [5]. Dopaminergic neuronal loss in the SNpc initiates the formation of Lewy bodies (LBs), which are composed α-synuclein (α-syn).
α-synuclein aggregation in the brain induces oxidative stress and neurodegeneration [6]. Elevated oxidative stress in the brain impairs cellular functions. The master antioxidant genes nuclear factor erythroid-related factor 2 (Nrf2) pathway, and its downstream targets, constitute an important role in oxidative stress. α-synuclein oligomerization and oxidative stress in the brain further activated glial cells, which released proinflammatory cytokines, such as nuclear transcription factor κB (NFκB), tumor necrosis factor-alpha (TNF-α), and interleukin 1 beta (IL-1β), which initiated neuroinflammation [7,8].
There are two main experimental animal models to study the pathophysiology of PD is toxin models and genetic models. The environmental toxin, 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP), induce acute animal models for PD. MPTP administration causes the death of neurons in the SNpc and striatum [9]. The MPTP-induced PD models provide a unique opportunity for researchers in studying the neuroprotective properties of novel drugs. MPTP in the brain is converted to toxic metabolite MPP+ by selectively inhibiting complex I of the mitochondrial electron transport chain. Mitochondrial dysfunction results in ATP disturbance, abnormal lipid and amino acid metabolism [10]. Anticholinergic drugs and other medications are used to treat PD. Natural substances obtained from medicinal plants are a favorite candidate for the prevention and management of PD models. These natural substances have been shown to inhibit protein misfolding and mitochondrial homeostasis. These drugs also showed antioxidative and anti-inflammatory effects [11]. Vitamins have also been reported to have antioxidative and anti-inflammatory properties, and are used for the treatment of various diseases [12,13]. Here, we used trolox (6-hydroxy-2,5,7,8-tetramethyl chroman-2-carboxylic acid), a water-soluble vitamin E, for the first time for the treatment of a MPTP-induced PD mouse model. Trolox has been considered a more potent free radical scavenger than its parental compound vitamin E [14]. Trolox showed antioxidative and anti-inflammatory properties in arthritic rat models [15]. Trolox prevented oxidative stress and inflammation in human and murine primary alveolar type II cells from injury [16]. Moreover, the neuroprotective potentials of Trolox have also been confirmed on hippocampal neurodegeneration in the ischemic model [17]. However, nobody has yet investigated their neuroprotective properties in PD models. The current study has anticipated, that Trolox may rescue mice's brains from MPTP-induced PD complications. We evaluated the neuroprotective effect of Trolox against MPTP-induced oxidative stress, neuroinflammation, and cognitive and motor dysfunction for the first time.

Trolox Inhibited MPTP-Induced Motor Dysfunction in PD
It has been reported that MPTP administration induced Parkinson's disease, and behavioral and motor dysfunctions in normal mice [18,19]. We also performed behavioral tests to investigate the effects of Trolox on motor functions in experimental animals. According to the open field test, the overall distance traveled by the MPTP-treated mice was significantly reduced in comparison to normal mice, whereas Trolox-treated mice greatly reversed this effect ( Figure 1a). Moreover, the MPTP-treated mice showed increased immobility time in comparison to saline-treated mice, but Trolox administration significantly reduced this effect (Figure 1b). In the pole test, the MPTP-treated mice demonstrated a substantial increase in the time taken to pivot and reach downward, whereas this impact was considerably diminished with Trolox administration (Figure 1c). The MPTP rodents revealed decreased holding time during the wire hang test, suggesting that the MPTP drug had a substantial negative impact on neuromuscular strength and has been increased with Trolox treatment (Figure 1d). Our findings showed that, Trolox significantly enhanced motor and cognitive functions in the MPTP-induced PD mouse model.

Trolox Treatment Reduced α-Synuclein Expression in Striatum and SNpc of MPTP-Induced PD Mice
Dopaminergic degradation in PD is related to the misfolding and aggregation of αsynuclein in the brain [20], and α-synuclein is overexpressed in MPTP-induced PD models. We also evaluated the effects of Trolox on α-synuclein expression, our western blot showed enhanced expression of α-synuclein in the brain of MPTP-induced PD, striatum, and SNpc; however, Trolox treatment significantly reduced this expression ( Figure 2a). Furthermore, we confirmed our results through confocal microscopy, which showed the increased immunoreactivity of α-synuclein in the striatum and SNpc of PD mice, and treatment with Trolox decreased the immunoreactivity of α-synuclein in the brain of PD mice (Figure 2b).

Trolox Treatment Reduced α-Synuclein Expression in Striatum and SNpc of MPTP-Induced PD Mice
Dopaminergic degradation in PD is related to the misfolding and aggregation of α-synuclein in the brain [20], and α-synuclein is overexpressed in MPTP-induced PD models. We also evaluated the effects of Trolox on α-synuclein expression, our western blot showed enhanced expression of α-synuclein in the brain of MPTP-induced PD, striatum, and SNpc; however, Trolox treatment significantly reduced this expression ( Figure 2a). Furthermore, we confirmed our results through confocal microscopy, which showed the increased immunoreactivity of α-synuclein in the striatum and SNpc of PD mice, and treatment with Trolox decreased the immunoreactivity of α-synuclein in the brain of PD mice (Figure 2b). (a) Expression of α-syn in the stri atum and SNpc of PD rodents, respectively (n = 5 mice/group), with β-actin used as a loading con trol; (b) the immunofluorescence findings of α-syn with their graphic representation. The values are expressed as the mean ± SD for indicated proteins. The values are given as the mean ± SD for the proteins specified (n = 10 mice/group), and there were three separate trials. The significant differ ences have been given in the graphs. * Significant difference between the control and MPTP group # significant difference between the MPTP group and Trolox-treated mouse. Significance: * p < 0.05 # p < 0.05.

Effects of Trolox on the Expression of Dopamine-Related Proteins in the Striatum and SNpc of the MPTP-Induced PD Mouse Model
Tyrosine hydroxylase (TH), vesicular monoamine transporter 2 (VMAT2), and dopamine transporter (DAT) are dopaminergic neuronal markers that are responsible for dopamine synthesis and transport. The loss of these proteins is involved in PD pathogenesis Our western blot results showed that the expressions of TH, VMAT2, and DAT were significantly reduced in the striatum and SNpc of the MPTP-induced PD mouse model in comparison to the saline-treated control mice, whereas the administration of Trolox significantly increased the expression of these markers (Figure 3a). We further confirmed our findings through immunostaining, which showed the decreased immunoreactivity of TH in the striatum and SNpc of the PD mouse models, while Trolox treatment substantially increased this expression (Figure 3b). We further evaluated the TH-positive neuron by using ImageJ software (NIH, Maryland MA, USA). Our findings suggested the decrease of TH-positive neurons in MPTP-treated rodents, while Trolox treatment increased them significantly (Figure 3c). (a) Expression of α-syn in the striatum and SNpc of PD rodents, respectively (n = 5 mice/group), with β-actin used as a loading control; (b) the immunofluorescence findings of α-syn with their graphic representation. The values are expressed as the mean ± SD for indicated proteins. The values are given as the mean ± SD for the proteins specified (n = 10 mice/group), and there were three separate trials. The significant differences have been given in the graphs. * Significant difference between the control and MPTP group; # significant difference between the MPTP group and Trolox-treated mouse. Significance: * p < 0.05; # p < 0.05.

Effects of Trolox on the Expression of Dopamine-Related Proteins in the Striatum and SNpc of the MPTP-Induced PD Mouse Model
Tyrosine hydroxylase (TH), vesicular monoamine transporter 2 (VMAT2), and dopamine transporter (DAT) are dopaminergic neuronal markers that are responsible for dopamine synthesis and transport. The loss of these proteins is involved in PD pathogenesis. Our western blot results showed that the expressions of TH, VMAT2, and DAT were significantly reduced in the striatum and SNpc of the MPTP-induced PD mouse model in comparison to the saline-treated control mice, whereas the administration of Trolox significantly increased the expression of these markers (Figure 3a). We further confirmed our findings through immunostaining, which showed the decreased immunoreactivity of TH in the striatum and SNpc of the PD mouse models, while Trolox treatment substantially increased this expression (Figure 3b). We further evaluated the TH-positive neuron by using ImageJ software (NIH, Maryland MA, USA). Our findings suggested the decrease of TH-positive neurons in MPTP-treated rodents, while Trolox treatment increased them significantly (Figure 3c). The values are given as the mean ± SD for the proteins specified (n = 10 mice/group), and there were three separate trials. The significant differences have been given in the graphs. * Significant difference between the control and MPTP group; # significant difference between the MPTP group and Trolox-treated mice. Significance: * p < 0.05; # p < 0.05.

Protective Role of Trolox against MPTP-Induced Oxidative Stress
Nuclear factor erythroid-related factor 2 (Nrf2), and its downstream gene heme oxygenase 1 (HO-1), play a major role in oxidative stress and neurodegeneration [21]. Impairments in the Nrf2/HO-1 pathway induced oxidative stress, reactive oxygen species (ROS) production, and mitochondrial dysfunction [22]. We also evaluated the antioxidative effect of Trolox on MPTP-induced oxidative stress in the mouse brain via the Nrf2/HO1 signaling pathway. Our immunoblot results showed a significantly decreased expression of nuclear Nrf2, and its target gene HO1, in the striatum and SNpc of MPTP-injected mice. Interestingly, Trolox treatment significantly upregulated nuclear Nrf2 and HO1 expression in the brain of PD mice. (Figure 4a). Furthermore, we performed ROS and LPO assay in the striatum and SNpc of MPTP-induced PD mice. Our results showed that ROS/LPO levels were significantly increased in the striatum and SNpc in PD mice brains, while Trolox treatment significantly downregulated ROS/LPO expression (Figure 4b,c). The overall finding suggested that Trolox can remarkably reduce oxidative stress in the mouse striatum and SNpc. The values are given as the mean ± SD for the proteins specified (n = 10 mice/group), and there were three separate trials. The significant differences have been given in the graphs. * Significant difference between the control and MPTP group; # significant difference between the MPTP group and Trolox-treated mice. Significance: * p < 0.05; # p < 0.05.

Protective Role of Trolox against MPTP-Induced Oxidative Stress
Nuclear factor erythroid-related factor 2 (Nrf2), and its downstream gene heme oxygenase 1 (HO-1), play a major role in oxidative stress and neurodegeneration [21]. Impairments in the Nrf2/HO-1 pathway induced oxidative stress, reactive oxygen species (ROS) production, and mitochondrial dysfunction [22]. We also evaluated the antioxidative effect of Trolox on MPTP-induced oxidative stress in the mouse brain via the Nrf2/HO1 signaling pathway. Our immunoblot results showed a significantly decreased expression of nuclear Nrf2, and its target gene HO1, in the striatum and SNpc of MPTP-injected mice. Interestingly, Trolox treatment significantly upregulated nuclear Nrf2 and HO1 expression in the brain of PD mice. (Figure 4a). Furthermore, we performed ROS and LPO assay in the striatum and SNpc of MPTP-induced PD mice. Our results showed that ROS/LPO levels were significantly increased in the striatum and SNpc in PD mice brains, while Trolox treatment significantly downregulated ROS/LPO expression (Figure 4b,c). The overall finding suggested that Trolox can remarkably reduce oxidative stress in the mouse striatum and SNpc. The values are given as the mean ± SD for the proteins specified ( 10 mice/group), and there were three separate trials. The significant differences have been given the graphs. * Significant difference between the control and MPTP group; # significant differe between the MPTP group and Trolox-treated mice. Significance: * p < 0.05; # p < 0.05.

Trolox Ameliorated MPTP-Induced Glial Cell Activation in PD Mice Brain
The MPTP-treated mice reported activated astrocytosis and microgliosis [23]. In PD brain, a small amount of α synuclein protein is released from neuronal cells, is tak by glial cells, and induces the expression of genes that are associated with immune fu tions [24]. Iba1 (ionized calcium-binding adaptor protein-1), and GFAP (glial fibrilla acidic protein) are two specific markers of activated microglial and astrocytic cells in brain, respectively [25]. We also evaluated the effects of Trolox on activated glial cells. O western blot results showed an increased expression of GFAP and Iba1 in MPTP-injec mice. However, this effect was significantly reversed by treatment with Trolox ( Figure 5 We confirmed our results further through confocal microscopy, which showed an creased immunoreactivity of GFAP and Iba1 in the striatum and SNpc of PD mouse mo els, while Trolox treatment substantially decreased this expression (Figure 5b,c). The values are given as the mean ± SD for the proteins specified (n = 10 mice/group), and there were three separate trials. The significant differences have been given in the graphs. * Significant difference between the control and MPTP group; # significant difference between the MPTP group and Trolox-treated mice. Significance: * p < 0.05; # p < 0.05.

Trolox Ameliorated MPTP-Induced Glial Cell Activation in PD Mice Brain
The MPTP-treated mice reported activated astrocytosis and microgliosis [23]. In the PD brain, a small amount of α synuclein protein is released from neuronal cells, is taken by glial cells, and induces the expression of genes that are associated with immune functions [24]. Iba1 (ionized calcium-binding adaptor protein-1), and GFAP (glial fibrillary acidic protein) are two specific markers of activated microglial and astrocytic cells in the brain, respectively [25]. We also evaluated the effects of Trolox on activated glial cells. Our western blot results showed an increased expression of GFAP and Iba1 in MPTP-injected mice. However, this effect was significantly reversed by treatment with Trolox (Figure 5a). We confirmed our results further through confocal microscopy, which showed an increased immunoreactivity of GFAP and Iba1 in the striatum and SNpc of PD mouse models, while Trolox treatment substantially decreased this expression (Figure 5b,c). . The values are given as the mean ± SD for the proteins specified (n = 10 mice/group), and there were three separate trials. The significant differences have been given in the graphs. * Significant difference between the control and MPTP group; # significant difference between the MPTP group and Trolox-treated mouse. Significance: * p < 0.05; # p < 0.05.

Trolox Attenuates MPTP-Induced Neuroinflammation in the Striatum and SNpc
The activated glial cells further release proinflammatory cytokines, which contributes to the etiology of PD. The nuclear factor-κB (NF-κB) is involved in various cellular processes and contributes to inflammatory responses. The phosphorylated NF-κB, plays a major role in neuroinflammation. It also activates the expression of other genes, such as tumor necrosis factor α (TNF-α) and interleukin-1β (IL-1β), which further induce neuroinflammation and neurodegeneration [26,27]. We also examined these inflammatory markers p-NFκB, TNF-α, and IL-1β by western blotting, and the results showed an increased expression of these markers in the striatum and SNpc of the MPTP-injected mice, while treatment with Trolox decreased the expression of these inflammatory markers in the PD mouse striatum and SNpc (Figure 6), suggesting that Trolox has a potent and effective role against MPTP-induced neuroinflammation. . The values are given as the mean ± SD for the proteins specified (n = 10 mice/group), and there were three separate trials. The significant differences have been given in the graphs. * Significant difference between the control and MPTP group; # significant difference between the MPTP group and Trolox-treated mouse. Significance: * p < 0.05; # p < 0.05.

Trolox Attenuates MPTP-Induced Neuroinflammation in the Striatum and SNpc
The activated glial cells further release proinflammatory cytokines, which contributes to the etiology of PD. The nuclear factor-κB (NF-κB) is involved in various cellular processes and contributes to inflammatory responses. The phosphorylated NF-κB, plays a major role in neuroinflammation. It also activates the expression of other genes, such as tumor necrosis factor α (TNF-α) and interleukin-1β (IL-1β), which further induce neuroinflammation and neurodegeneration [26,27]. We also examined these inflammatory markers p-NFκB, TNF-α, and IL-1β by western blotting, and the results showed an increased expression of these markers in the striatum and SNpc of the MPTP-injected mice, while treatment with Trolox decreased the expression of these inflammatory markers in the PD mouse striatum and SNpc (Figure 6), suggesting that Trolox has a potent and effective role against MPTPinduced neuroinflammation. Figure 6. Neuroprotective role of Trolox against MPTP-induced neuroinflammation. The immun lot outcomes of p-NFκB, TNF-α, and IL-1β in the striatum and SNpc of PD mice, and their grap representation, of the different experimental groups. β-actin served as a loading control. The val are given as the mean ± SD for the proteins specified (n = 10 mice/group), and there were th separate trials. The significant differences have been given in the graphs. * Significant differe between the control and MPTP group; # significant difference between the MPTP group and Tro treated mouse. Significance: * p < 0.05; # p < 0.05.

Discussion
Parkinson's disease (PD) is the most prevalent and complicated neurodegenerat disorder [28]. The pathological features of PD include synucleinopathy, oxidative stre loss of the dopamine-related protein, neuroinflammation, and motor dysfunction. The fore, in the current study, we focused on these factors and used Trolox to target th pathological elements. This prevailing study provides the first direct evidence that Tro prevented the MPTP-induced PD mice model. To demonstrate the positive impact Trolox, we assessed its effect on behavior alterations and our findings showed that Tro treatment restored behavioral alterations in the MPTP-induced PD mice model. Previo studies have also shown that MPTP administration caused the production and deposit of α-synuclein in the brain of animals [29][30][31]. Our results showed that Trolox treatme significantly decreased α-synuclein overexpression caused by MPTP injection. Tyros hydroxylase (TH), vesicular monoamine transporter 2 (VMAT2), and dopamine tra porter (DAT) are the dopaminergic neuronal markers responsible for dopamine format in the brain. A previous study has shown that a reduction in TH, VMAT2, and DAT lea to dopamine depletion in substantial nigra of Parkinson's disease patients. Furthermo it is also involved in the loss of dopaminergic neurons, which is the main pathologi Figure 6. Neuroprotective role of Trolox against MPTP-induced neuroinflammation. The immunoblot outcomes of p-NFκB, TNF-α, and IL-1β in the striatum and SNpc of PD mice, and their graphic representation, of the different experimental groups. β-actin served as a loading control. The values are given as the mean ± SD for the proteins specified (n = 10 mice/group), and there were three separate trials. The significant differences have been given in the graphs. * Significant difference between the control and MPTP group; # significant difference between the MPTP group and Troloxtreated mouse. Significance: * p < 0.05; # p < 0.05.

Discussion
Parkinson's disease (PD) is the most prevalent and complicated neurodegenerative disorder [28]. The pathological features of PD include synucleinopathy, oxidative stress, loss of the dopamine-related protein, neuroinflammation, and motor dysfunction. Therefore, in the current study, we focused on these factors and used Trolox to target these pathological elements. This prevailing study provides the first direct evidence that Trolox prevented the MPTP-induced PD mice model. To demonstrate the positive impact of Trolox, we assessed its effect on behavior alterations and our findings showed that Trolox treatment restored behavioral alterations in the MPTP-induced PD mice model. Previous studies have also shown that MPTP administration caused the production and deposition of α-synuclein in the brain of animals [29][30][31]. Our results showed that Trolox treatments significantly decreased α-synuclein overexpression caused by MPTP injection. Tyrosine hydroxylase (TH), vesicular monoamine transporter 2 (VMAT2), and dopamine transporter (DAT) are the dopaminergic neuronal markers responsible for dopamine formation in the brain. A previous study has shown that a reduction in TH, VMAT2, and DAT leads to dopamine depletion in substantial nigra of Parkinson's disease patients. Furthermore, it is also involved in the loss of dopaminergic neurons, which is the main pathological hallmark of PD [32,33]. Following this research, our results suggested a decrease of TH, VMAT2, and DAT in the MPTP mouse, although Trolox administration substantially reversed this action by uplifting the expression of these markers.
Oxidative stress is also involved in the degeneration of dopaminergic neurons and the pathogenesis of PD. Nuclear factor erythroid-2-related factor 2 (Nrf2) is a transcription factor that activates several protective genes including heme oxygenase-1 (HO1), which further protects cells from oxidative stress and neurodegeneration. A series of studies reported that bioactive compounds showed the properties to activate the Nrf2/HO1 pathways and ameliorate PD-related neurotoxicity [34][35][36]. We also examined the expression of Nrf2/HO1 in the brains of experimental mice. Collectively, our results suggested that trolox has strong agonistic effects against the reduced expression of Nrf2 and HO1 in the MPTP-induced PD mice brains. Enhanced lipid peroxidation (LPO) and reactive oxygen species (ROS) are also related to elevated oxidative stress [37]. Therefore, we also performed ROS and LPO assays, which showed that Trolox treatment reduced ROS and LPO levels.
Astrocytes and microglia regulate synapse formation, phagocytosis, and homeostasis in the brain [38]. They also play a critical role in the neuroprotection and regeneration of the injured brain [39]. They also respond rapidly to reactive oxygen species (ROS), and inhibit neuroinflammation. The overactivation of the glial cells induces neuroinflammation [40]. Here, we observed that Trolox administration prominently reduced Iba1 and GFAP expression, which were elevated with MPTP injection. Activated glial cells and injured neurons further release inflammatory mediators such as nuclear factor-κB (p-NFκB), tumor necrosis factor-α (TNF-α), and interleukin 1-beta (IL-1β). The release of these mediators plays a major role in the development of neuroinflammation [41]. Our results also showed a higher expression of these inflammatory markers in the striatum and SNpc of MPTP-injected mice, while the treatment with Trolox significantly decreased the expression of these inflammatory markers.
The proposed mechanism of action of trolox is given in Figure 7. factor that activates several protective genes including heme oxygenase-1 (HO1), which further protects cells from oxidative stress and neurodegeneration. A series of studies reported that bioactive compounds showed the properties to activate the Nrf2/HO1 pathways and ameliorate PD-related neurotoxicity [34,35,36]. We also examined the expression of Nrf2/HO1 in the brains of experimental mice. Collectively, our results suggested that trolox has strong agonistic effects against the reduced expression of Nrf2 and HO1 in the MPTP-induced PD mice brains. Enhanced lipid peroxidation (LPO) and reactive oxygen species (ROS) are also related to elevated oxidative stress [37]. Therefore, we also performed ROS and LPO assays, which showed that Trolox treatment reduced ROS and LPO levels.
Astrocytes and microglia regulate synapse formation, phagocytosis, and homeostasis in the brain [38]. They also play a critical role in the neuroprotection and regeneration of the injured brain [39]. They also respond rapidly to reactive oxygen species (ROS), and inhibit neuroinflammation. The overactivation of the glial cells induces neuroinflammation [40]. Here, we observed that Trolox administration prominently reduced Iba1 and GFAP expression, which were elevated with MPTP injection. Activated glial cells and injured neurons further release inflammatory mediators such as nuclear factor-κB (p-NFκB), tumor necrosis factor-α (TNF-α), and interleukin 1-beta (IL-1β). The release of these mediators plays a major role in the development of neuroinflammation [41]. Our results also showed a higher expression of these inflammatory markers in the striatum and SNpc of MPTP-injected mice, while the treatment with Trolox significantly decreased the expression of these inflammatory markers.
The proposed mechanism of action of trolox is given in Figure 7.

Animal Groupings and Drug Administration
Male wild-type (WT) C5BL/6N mice, eight weeks old and of an average weight of 25-30 g, were purchased from Samtako Bio labs (Ulsan, Republic of Korea). All mice were acclimatized for one week. They were housed at room temperature (25 • C) and were provided a 12 h dark/light cycle with 60 ± 10% humidity. After acclimating, the mice were divided into four groups (n = 10 mice per group): (1) a control group (receiving 0.9% saline); (2) an MPTP-treated group (MPTP was injected at a dose of 30 mg/kg intravenously for 5 days), as previously used [42]; (3) a Trolox-treated group (intraperitoneally treated with Trolox at a dose of 50 mg/kg for 10 days); (4) The sham-treated group (Trolox 50 mg/kg i.p for 10 days) ( Figure 8). All the experiments involving animals were implemented in agreement with guidelines and principles approved by the Institutional Animal Care and Use Committee (IACUC), Division of Applied Life Sciences, Gyeongsang National University, Republic of Korea (Approval ID: 125).

Animal Groupings and Drug Administration
Male wild-type (WT) C5BL/6N mice, eight weeks old and of an average weight of 25-30 g, were purchased from Samtako Bio labs (Ulsan, Republic of Korea). All mice were acclimatized for one week. They were housed at room temperature (25 °C) and were provided a 12 h dark/light cycle with 60 ± 10% humidity. After acclimating, the mice were divided into four groups (n = 10 mice per group): (1) a control group (receiving 0.9% saline); (2) an MPTP-treated group (MPTP was injected at a dose of 30 mg/kg intravenously for 5 days), as previously used [42]; (3)

Open Field Test
The spontaneous movement activity was assessed by using an open field setup (40 × 40 cm in diameter, 40 cm in height) [42]. Video tracking software 3.0 (Panlab, Barcelona, Spain) was employed to record the mice's movements as they traveled through the 16 evenly sized squares constituting an open field box. Before the trial, the mice were given a few hours to adapt to new surroundings. Each mouse is individually put in the same corner. The open area was cleaned with an ethanol solution (10%) and dried to eliminate smells between each test. The mice's total travel distance and time spent in the center were assessed. The duration is indicated in seconds, whereas the distance is specified in centimeters.

Pole Test
A wooden pole with a diameter of 10 mm and a height of 40 cm constituted the pole test apparatus. Mice were placed head up near the summit of a wooden pole with a rough surface and the duration to reach the level was examined. The test was administered three times, with each trial lasting 10 min. To evaluate the behavioral changes, the mean of three descending times was employed.

Wire Hang Test
The wire hang test, also known as the traction test, was used to evaluate neuromuscular strength [43]. The mice were adapted to the behavioral chamber for 20-30 min before the test. Mice were supported by their tails and slickly positioned on a horizontally elongated wire and supported until they gripped the wire by using their fore and hind paws, as earlier reported [35]. The wire was fixed 20 cm above the ground to deter falls but not inflict any harm in the case of falling. The experiment was conducted ten times, and the average results were assessed. After each test, the rodents were allowed to recuperate. The results are displayed as "latency to fall to the earth" in seconds.

Extraction of the Protein from Mouse Brain
After the behavior analysis, the mice were sacrificed, the brain tissue was extracted carefully, and the striatum and substantia nigra pars compacta (SNpc) regions were separated. The striatum and SNpc were individually homogenized in Pro-Prep TM protein extraction solution (iNtRON Biotechnology, Sungnam, Republic of Korea), and centrifuged for 25 min at 13,000 rpm and 4 • C. The supernatants were collected and kept at −70 • C.

Western Blot Analysis
Western blot analysis was carried out, as previously described [44][45][46]. Briefly, a Bio-Rad protein assay tool (Bio-Rad Laboratories, Hercules, CA, USA) was used to assess the protein concentrations. Proteins in equal quantities (20 µg) were electrophoresed on 10-20% BoltTM Mini Gels and separated on 0.2-µm PVDF Immobilon membranes (Merck Millipore, MA, USA). The membranes were blocked in 5% (w/v) skimmed milk before being incubated with primary antibodies (1:1000 dilution) overnight at 4 • C to minimize nonspecific binding. The membrane was washed and processed with HRP-conjugated antimouse (W402B) Promega (Madison, WI, USA) secondary antibodies after being incubated with primary antibodies. The particular bands were analyzed by EzWestLumiOne (ATTO, Tokyo, Japan).
Computer-based ImageJ software (NIH, Maryland MA, USA) was used to measure the band densities, and the graphs were generated with GraphPad Prism 8 software (San Diego, CA, USA).

Lipid Peroxidation (LPO) Assay
LPO was performed to assess oxidative stress, as previously described [47,48]. In brief, the striatum and SNpc regions' tissue homogenate was evaluated using a lipid peroxidation (MDA) colorimetric/fluorometric assay reagent to assess a marker of LPOfree malondialdehyde (MDA) according to the manufacturer's instructions and prior work by our team, respectively.

Reactive Oxygen Species (ROS) Assay
A ROS assay was conducted, as previously described [37,49]. The ROS assay relies on the conversion of 2 ,7 -dichlorodihydrofluorescein diacetate (DCFH-DA) to 2 ,7dichlorofluorescein (DCF). To achieve the final concentration of 2.5 mg tissue/500 L, the striatum and SNpc brain homogenates from the various groups were diluted with icecold Lock's buffer at a 1:20 ratio. Then, to create fluorescent DCF from DCFH-DA, 1 mL of Locke's buffer combination (pH ± 7.4), 0.2 mL of homogenates, and 10 mL of 5 mM 7-dichlorodihydrofluorescein diacetate (DCFH-DA) were incubated for 15 min at room temperature. A microplate scanner was used to measure the formation of DCF from DCFH-DA at 484 nm (excitation) and 530 nm (emission). We measured parallel blanks in the absence of homogenate to enable the calculation of DCF formation (background fluorescence). The values were presented as DCF-formed pmol/protein mg.

Immunofluorescence Staining
After the completion of the treatments and behavioral analysis, the mice (n = 5 per group) were deeply anesthetized and perfused with normal saline solution (0.9%) and 4% cold paraformaldehyde. The brain samples were immediately separated and fixed with paraformaldehyde for 72 h; then, all the brains were transferred to 20% sucrose for 72 h, as described previously [50,51]. The brain tissues were placed in optimal cutting temperature compound (OCT) (Sakura Finetek Japan Co., Ltd., Tokyo, Japan), to sustain the appropriate cutting temperature, using a cryo-microtome ((Leica cryostat CM 3050, Germany). The brain samples were sequentially snipped into 14 µm thick coronal sections. Following a 10 min rehydration with phosphate-buffered saline (PBS), the brain tissues were then treated for 5 min with proteinase K, rinsed with PBS (0.01 mM), and blocked with 2% goat normal serum in PBS containing 0.1% Triton X-100 for 50 minutes. After blocking, the slides were treated with primary antibodies overnight at 4 • C. Slides were then treated with secondary antibodies labeled with fluorescein isothiocyanate (FITC) for 90 min at room temperature. The slides were mounted with DPX (distyrene plasticizer xylene) mounting medium and covered with glass coverslips, according to previously described protocols [52][53][54]. Images were taken using a confocal scanning microscope (FluoView FV1000; Olympus, Tokyo, Japan). The ImageJ software (version 1.50, NIH, 1 March 2016; https://imagej.nih.gov/ij/, Bethesda, MD, USA) was used for further evaluation and, for graph plotting, PRISM8 software was used.

Statistical Analysis
The X-ray films were scanned, and ImageJ software was used to analyze the density of bands. The densities were represented in terms of the mean and standard error of the mean (SEM). To compare the various groups, a one-way ANOVA (analysis of variance) was used, followed by independent Student t-tests and Tukey's multiple comparison analysis. The graphs have been generated by employing the Prism8 softwaree (GraphPad Software, Inc., San Diego, CA, USA). p values < 0.05 were deemed statistically significant between the different treated groups.

Conclusions
In conclusion, our findings showed evidence that Trolox can potentially protect the brain from MPTP-induced PD mice. Trolox treatment decreases the alpha-synuclein expression in PD mice and increases the TH, DAT, and VMAT expression. Further Trolox treatment activated the Nrf2/HO1 pathways and ameliorated PD-related neurotoxicity. Trolox administration also reduced glial cell expression, which was elevated with MPTP injection. Other inflammatory mediators, such as p-NFκB, TNF-α, and IL1-β, were also reduced with Trolox treatment. Therefore, collectively, Trolox treatment protects the PD mice from MPTP-induced neuronal loss, oxidative stress, neuroinflammation, and motor dysfunction.